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Human ABCB1 with an ABCB11-like degenerate nucleotide binding site maintains transport activity by avoiding nucleotide occlusion

Several ABC exporters carry a degenerate nucleotide binding site (NBS) that is unable to hydrolyze ATP at a rate sufficient for sustaining transport activity. A hallmark of a degenerate NBS is the lack of the catalytic glutamate in the Walker B motif in the nucleotide binding domain (NBD). The multi...

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Published in:PLoS genetics 2020-10, Vol.16 (10), p.e1009016-e1009016
Main Authors: Goda, Katalin, Dönmez-Cakil, Yaprak, Tarapcsák, Szabolcs, Szalóki, Gábor, Szöllősi, Dániel, Parveen, Zahida, Türk, Dóra, Szakács, Gergely, Chiba, Peter, Stockner, Thomas
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cited_by cdi_FETCH-LOGICAL-c726t-74002516f48516cdd82b3afc78af5a0876e2d449e8bc841bdc25b551d867848c3
cites cdi_FETCH-LOGICAL-c726t-74002516f48516cdd82b3afc78af5a0876e2d449e8bc841bdc25b551d867848c3
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container_issue 10
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container_title PLoS genetics
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creator Goda, Katalin
Dönmez-Cakil, Yaprak
Tarapcsák, Szabolcs
Szalóki, Gábor
Szöllősi, Dániel
Parveen, Zahida
Türk, Dóra
Szakács, Gergely
Chiba, Peter
Stockner, Thomas
description Several ABC exporters carry a degenerate nucleotide binding site (NBS) that is unable to hydrolyze ATP at a rate sufficient for sustaining transport activity. A hallmark of a degenerate NBS is the lack of the catalytic glutamate in the Walker B motif in the nucleotide binding domain (NBD). The multidrug resistance transporter ABCB1 (P-glycoprotein) has two canonical NBSs, and mutation of the catalytic glutamate E556 in NBS1 renders ABCB1 transport-incompetent. In contrast, the closely related bile salt export pump ABCB11 (BSEP), which shares 49% sequence identity with ABCB1, naturally contains a methionine in place of the catalytic glutamate. The NBD-NBD interfaces of ABCB1 and ABCB11 differ only in four residues, all within NBS1. Mutation of the catalytic glutamate in ABCB1 results in the occlusion of ATP in NBS1, leading to the arrest of the transport cycle. Here we show that despite the catalytic glutamate mutation (E556M), ABCB1 regains its ATP-dependent transport activity, when three additional diverging residues are also replaced. Molecular dynamics simulations revealed that the rescue of ATPase activity is due to the modified geometry of NBS1, resulting in a weaker interaction with ATP, which allows the quadruple mutant to evade the conformationally locked pre-hydrolytic state to proceed to ATP-driven transport. In summary, we show that ABCB1 can be transformed into an active transporter with only one functional catalytic site by preventing the formation of the ATP-locked pre-hydrolytic state in the non-canonical site.
doi_str_mv 10.1371/journal.pgen.1009016
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A hallmark of a degenerate NBS is the lack of the catalytic glutamate in the Walker B motif in the nucleotide binding domain (NBD). The multidrug resistance transporter ABCB1 (P-glycoprotein) has two canonical NBSs, and mutation of the catalytic glutamate E556 in NBS1 renders ABCB1 transport-incompetent. In contrast, the closely related bile salt export pump ABCB11 (BSEP), which shares 49% sequence identity with ABCB1, naturally contains a methionine in place of the catalytic glutamate. The NBD-NBD interfaces of ABCB1 and ABCB11 differ only in four residues, all within NBS1. Mutation of the catalytic glutamate in ABCB1 results in the occlusion of ATP in NBS1, leading to the arrest of the transport cycle. Here we show that despite the catalytic glutamate mutation (E556M), ABCB1 regains its ATP-dependent transport activity, when three additional diverging residues are also replaced. Molecular dynamics simulations revealed that the rescue of ATPase activity is due to the modified geometry of NBS1, resulting in a weaker interaction with ATP, which allows the quadruple mutant to evade the conformationally locked pre-hydrolytic state to proceed to ATP-driven transport. In summary, we show that ABCB1 can be transformed into an active transporter with only one functional catalytic site by preventing the formation of the ATP-locked pre-hydrolytic state in the non-canonical site.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>33031417</pmid><doi>10.1371/journal.pgen.1009016</doi><orcidid>https://orcid.org/0000-0003-4641-3065</orcidid><orcidid>https://orcid.org/0000-0001-7182-0135</orcidid><orcidid>https://orcid.org/0000-0002-4605-1167</orcidid><orcidid>https://orcid.org/0000-0002-9311-7827</orcidid><orcidid>https://orcid.org/0000-0001-6190-9128</orcidid><orcidid>https://orcid.org/0000-0003-1215-3453</orcidid><orcidid>https://orcid.org/0000-0003-2001-7400</orcidid><oa>free_for_read</oa></addata></record>
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subjects AAA Domain - genetics
ABC transporters
Adenosine triphosphatase
Adenosine Triphosphate - genetics
Amino Acid Sequence
Amino acids
ATP Binding Cassette Transporter, Subfamily B - genetics
ATP Binding Cassette Transporter, Subfamily B, Member 11 - genetics
Bile
Binding sites
Binding sites (Biochemistry)
Binding Sites - genetics
Biological Transport - genetics
Biological Transport, Active - genetics
Biology
Biology and Life Sciences
Biophysics
Catalytic Domain - genetics
Cell Cycle Proteins - genetics
Funding
Genetic aspects
Glutamic Acid - genetics
Glycoproteins
Humans
Hydrolysis
Interfaces
Medical research
Medicine
Methionine
Methionine - genetics
Molecular Dynamics Simulation
Multidrug resistance
Mutation
Mutation - genetics
Nuclear Proteins - genetics
Nucleotides
Nucleotides - genetics
Occlusion
P-Glycoprotein
Pharmacology
Physical sciences
Physiological aspects
Physiology
Protein Binding - genetics
Protein Domains - genetics
Proteins
Research and analysis methods
Supervision
title Human ABCB1 with an ABCB11-like degenerate nucleotide binding site maintains transport activity by avoiding nucleotide occlusion
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